Sung Ho Yang

Mussel-inspired encapsulation and functionalization of individual yeast cells.

The individual encapsulation of living cells has a great impact on the area of cell-based sensors and devices as well as fundamental studies in cell biology. In this work, living yeast cells were individually encapsulated with functionalizable, artificial polydopamine shells, inspired by an adhesive protein in mussels. Yeast cells maintained their viability within polydopamine, and the cell cycle was controlled by the thickness of the shells. In addition, the artificial shells aided the cell in offering much stronger resistance against foreign aggression, such as lyticase. After formation of the polydopamine shells, the shells were functionalized with streptavidin by utilizing the chemical reactivity of polydopamine, and the functionalized cells were biospecifically immobilized onto the defined surfaces. Our work suggests a biomimetic approach to the encapsulation and functionalization of individual living cells with covalently bonded, artificial shells.

Bioinspired Functionalization of Silica-Encapsulated Yeast Cells†

Cell-surface modification is usually achieved by sophisticated but complicated methods, such as the introduction of nonbiogenic functional groups by metabolic or genetic engineering. Although such methods have evolved into biocompatible and bioorthogonal strategies, the possibility that the direct insertion of functional moieties causes significant perturbations to cell membranes still remains. For a decade, encapsulation methods have been developed as an alternative, indirect approach to cell-surface modifications, as it is thought that the cell integrity would not be perturbed by the encapsulation methods where functional moieties are introduced onto the cell surface without any direct contact with cell membranes. For example, the noncovalent adsorption of macromolecules, mostly by layer-by-layer (LbL) processes, has been utilized to introduce various functionalities, including fluorescent and magnetic properties, catalytic moieties, and supporting templates, to the living cells. On the other hand, recently reported artificial shells, which robustly encapsulate individual living cells, have attracted a great deal of attention as a new approach to cell-surface modifications and formation of artificial spores, because the artificial shells were reported to enhance cell viability and also to control cell division; these factors would be beneficial in the development of biosensor circuits, lab-ona-chip systems, and bioreactors, as well as for fundamental studies in cell biology. It is therefore anticipated that the synergistic combination of the protective encapsulation and the cell-surface functionalization would make a significant step towards the aforementioned applications. Despite the advantages of physically protective shells, the utilization of the artificial shells for practical applications still remains a challenge. The mechanical robustness and chemical inertness of the artificial shells prove beneficial for protecting living cells, but, contradictorily, these properties limit chemical functionalizations of the shells in terms of reactivity. For example, calcium carbonate or calcium phosphate shells lack chemical reactivity. Although the chemistry of silicon is well established, the functionalization of silica shells requires harsh conditions, such as high pH values and harmful solvents. Therefore, it is a prerequisite for any application that the functionalizabilty of the artificial shells is ensured along with the mechanical robustness of the protective shells. Herein we report a bioinspired method for the encapsulation of individual living yeast cells with functionalizable silica shells. Specifically, we used biomimetic silicification, which was inspired by the biosilicification of diatoms. Biomimetic silicification is achieved by specific interactions between silicic acid derivatives and cationic polyamines, such as natural and synthetic peptides, and synthetic polymers: the self-assembled structure of polyamines is thought to act as a catalytic template for the in vivo polycondensation of silicic acid derivatives. We reasoned that chemical functional groups would be introduced directly to the biomimetically formed silica by adding silanol derivatives that contain functional groups in the course of biomimetic polycondensation of silicic acid derivatives. (3-Mercaptopropyl)trimethoxysilane (MPTMS) was selected as a model additive because it was reported to be polycondensed simultaneously with silicic acid under physiologically mild conditions. 12] The functionalizable silica shells formed in this work would expand the utility of artificial shells, because the thiol group in the silica shell can be used for introducing various functions through specific reactions of the thiol moiety with maleimide derivatives under biocompatible conditions (aqueous solution, pH 7.4; Figure 1). The polyelectrolyte multilayer of poly(ethyleneimine) (PEI, Mw: 750 000) and poly(sodium 4-styrenesulfonate) (PSS, Mw: 70000) was used as a catalytic template for biomimetic silicification because previous studies indicated that PEI was biocompatible and acts as a catalyst for biomimetic silica formation. PEI and PSS were alternately deposited onto the surface of Saccharomyces cerevisiae (S. cerevisiae ; baker s yeast). The layer-by-layer processes were initiated with PEI so that electrostatic interactions occur with the negatively charged cell surfaces, and terminated with PEI so that catalytic interactions occur with silicic acid derivatives at the outer interface. For the individual encapsulation of yeast cells with thiol-functionalized silica (SiO2 ; i.e., formation of yeast@SiO2 ), the PEI/PSS multilayercoated cells were placed for 30 min in a silicic acid derivative solution (100 mm), which had been prepared by adding [*] Dr. S. H. Yang, E. H. Ko, Prof. Dr. I. S. Choi Molecular-Level Interface Research Center Department of Chemistry, KAIST, Daejeon 305-701 (Korea) Fax: (+ 82)42-350-2810 E-mail: ischoi@kaist.ac.kr Homepage: http://cisgroup.kaist.ac.kr

Interfacing Living Yeast Cells with Graphene Oxide Nanosheaths

The first example of the encapsulation of living yeast cells with multilayers of GO nanosheets via LbL self-assembly is reported. The GO nanosheets with opposite charges are alternatively coated onto the individual yeast cells while preserving the viability of the yeast cells, thus affording a means of interfacing graphene with living yeast cells. This approach is expanded by integrating other organic polymers or inorganic nanoparticles to the cells by hybridizing the entries with GO nanosheets through LbL self-assembly. It is demonstrated that incorporated iron oxide nanoparticles can deliver magnetic properties to the biological systems, allowing the integration of new physical and chemical functions for living cells with a combination of GO nanosheets.

Cytocompatible Encapsulation of Individual Chlorella Cells within Titanium Dioxide Shells by a Designed Catalytic Peptide

The individual encapsulation of living cells has a great impact on the areas of single cell-based sensors and devices as well as fundamental studies in single cell-based biology. In this work, living Chlorella cells were encapsulated individually with abiological, functionalizable TiO(2), by a designed catalytic peptide that was inspired by biosilicification of diatoms in nature. The bioinspired cytocompatible reaction conditions allowed the encapsulated Chlorella cells to maintain their viability and original shapes. After formation of the TiO(2) shells, the shells were postfunctionalized by using catechol chemistry. Our work suggests a bioinspired approach to the interfacing of individual living cells with abiological materials in a controlled manner.

Counteranion-Directed, Biomimetic Control of Silica Nanostructures on Surfaces Inspired by Biosilicification Found in Diatoms†

Films of several biogenic and nonbiogenic inorganicspecies have already been synthesized under mild conditions(ambient pressure, room temperature or below, and near-neutralpHvalues)thatmimicthephysiologicalconditionsusedinnature.However,structuralcontroloftheinorganicfilmsstillremains challenging in the biomimetic approach.The nanometer-scale, spatial control of silica structures onsurfaces has many potential applications, such as heteroge-neous catalysis,

Bio-inspired silicification on patterned surfaces generated by microcontact printing and layer-by-layer self-assembly.

Micropatterns of silica were generated under biocompatible conditions by a combination of microcontact printing (muCP), layer-by-layer (LbL) self-assembly, and biomimetic silicification. Quaternary amine-containing poly(diallyl dimethyl ammonium chloride) induced polycondensation of silicic acid, resulting in spatioselective formation of silica micropatterns. Scale bar: 10 microm.

Biomimetic Approach to the Formation of Titanium Dioxide Thin Films by Using Poly(2-(dimethylamino)ethyl methacrylate)

We demonstrate that the biomimetic method-which has been used for the formation of silica thin films-also could be applied directly to the formation of titanium dioxide (TiO(2)) thin films, which are technologically important materials because of their applications to photocatalytic purifiers, photochemical solar cells, and others. After generation of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) films on gold surfaces by surface-initiated polymerization, titanium bis(ammonium lactato)dihydroxide was used as a precursor of TiO(2). The TiO(2)/PDMAEMA films were successfully formed on the surfaces in aqueous solution at neutral pH (pH 6.7) and room temperature, and were characterized by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, atomic force microscopy, scanning electron microscopy, and X-ray diffractometry. The formed TiO(2) films have an amorphous nature and large area uniformity in thickness. The degree of crystallization was controlled by annealing. We also investigated the pH effect and the phosphate incorporation in the films by using phosphate-buffered solutions. The TiO(2) films were formed in all the employed pH values in the range of 2 to 12, but phosphate anions were found to be incorporated into the films facilely only at low pH.

Structure Modulation of Silica Microspheres in Bio-Inspired Silicification: Effects of TEOS Concentration

Silica nanoand microspheres have applications in various areas, such as in photonic crystals, catalysis, biosensors, bioassay, and drug delivery. Numerous synthetic methods have been developed and been modified to meet the demands of the applications mentioned above. In particular, the Stcber method is considered as a basic platform for the chemical synthesis of silica spheres: it generally uses ammonia as a catalyst and silicon alkoxide as a precursor in the water/ethanol co-solvent system. Although the conventional Stcber method is quite useful for sizeand shape-control of silica structures, the reaction conditions are harsh owing to the use of ammonia (pH>12) and cannot be applied to biological systems, such as living cells. In this respect, biomimetic (or bio-inspired) silicification, inspired by diatom and glass sponges, was suggested as an alternative approach for the mild and biocompatible formation of silica structures, because it proceeds under physiologically mild conditions (i.e., neutral pH, room temperature, and ambient pressure). The bio-inspired silicification has successfully been applied to the coating of individual living cells without deterioration of cell viability by us. We also have recently reported that individually separated silica microspheres were formed under relatively mild conditions in the presence of cetyltrimethylammonium bromide (CTAB) by using cysteamine (HSCH2CH2NH2) as a biomimetic hydrolysis catalyst, inspired by silicatein, a silica-forming protein, found in glass sponge. It is noteworthy that the diameter of the formed silica spheres was on the micrometer-scale, because microspheres had barely been found in both Stcber and modified Stcber methods. Understanding how the bioinspired silicification was affected by reaction parameters in the cysteamine/CTAB system, such as reactant concentrations and solvents, was required for the detailed elucidation of mechanisms and the morphological control of silica. However, there have been few reports on the effects of the concetrations of silica precursors on silica morphogenesis. Herein, we systematically investigated the effects of the concentration of tetraethyl orthosilicate (TEOS) in detail, along with the ratio of water and ethanol. The synthetic procedure was as follows (Figure 1). The final concentrations of cysteamine and CTAB were fixed to be 50 mm and 5 mm, respectively, after optimization for silica formation. We varied the water/ethanol ratio from 0.6:1 to 1:1 (v/v), and the concentration of TEOS from 80 to 140 mm in 20 mm-intervals. Although the observable changes in the reaction could be seen after 45 minutes, the silicification was performed for 3 hours for comparative studies. The resulting silica precipitates were washed with ethanol several times using centrifugation, dispersed in ethanol, and characterized by attenuated total reflectance infrared (ATR-IR) spectroscopy and field-emission scanning electron microscopy (FE-SEM). The IR spectra showed the characteristic peaks at 1049 (Si O Si asymmetric stretching), 955 (Si O stretching), and 784 cm 1 (Si O Si symmetric stretching) after silicification (for the representative IR spectrum, see the Supporting Information, Figure S1). To investigate the effects of TEOS concentration, we first varied only the concentration of TEOS in the 0.65:1 water/ ethanol system, while keeping the other parameters (the concentrations of cysteamine and CTAB) the same. Of interest, the silica morphology was found to be affected greatly by the concentration of TEOS, as shown in the FE-SEM micrographs (Figure 2a). Specifically, interconnected aggre[a] J. H. Park, J. Y. Choi, T. Park, S. H. Yang, S. Kwon, Prof. Dr. H.-S. Lee, Prof. Dr. I. S. Choi Molecular-Level Interface Research Center Department of Chemistry KAIST Daejeon 305-701 (Korea) Fax: (+82)42-350-2810 E-mail : ischoi@kaist.ac.kr hee-seung_lee@kaist.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201100265.

Rosette-shaped calcite structures at surfaces: mechanistic implications for CaCO3 crystallization.

Biomineralization is believed to be achieved by the intimate cooperation of soluble macromolecules and an insoluble matrix at the specific inorganic-organic interface. It has been reported that positively charged matrices play an important role in controlling the structure of CaCO(3) at surfaces, although detailed mechanisms remain unclear. In this work, we studied the transformation from amorphous CaCO(3) to calcite crystals on surfaces by using thin films of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) and its quaternized form. The positively charged PDMAEMA film was found to possess unique properties for CaCO(3) crystallization: individually separated, single calcite crystals were formed on the PDMAEMA film in the absence of poly(acrylic acid) (PAA), while circularly fused calcite crystals were formed in the presence of PAA. The circularly fused (rosette-shaped) calcite crystals could be changed from a completely packed rosette to a ring-shaped, hollow structure by tuning the crystallization conditions. A number of factors, such as reaction time, amount of (NH(4))(2)CO(3), concentration of PAA, and charge of matrix-films, were varied systematically, and we now propose a mechanism based on these observations.